Molecular imaging continues to evolve into a discipline in its own right with applications to many areas of biology and clinical medicine. The long-term goal of this first-time competitive renewal application is to develop technologies focused on reporter genes for use with PET, optical, and other emerging modalities for applications to gene therapy, cancer biology, developmental biology, as well as many other areas. The general hypothesis is that reporter gene technologies can be developed and utilized to study fundamental molecular/cellular events in living subjects. Excellent progress has been made in this regard over the last 4 year funding period. We have helped to push the field forward by developing and validating enzyme based (Herpes-simplex Virus Type 1 thymidine kinase (HSVI-tk)) and receptor based (Dopamine Type 2 receptor (D2R)) reporter gene assays. We have applied these reporter genes primarily in the area of imaging gene therapy. The specific aims of the current renewal are to take the developed approaches to the next level by providing tools "systems imaging" by imaging intracellular networks in living subjects. Protein-protein interactions are at the heart of many biological process including normal and diseased tissue. In Aim 1 we refine a recently developed strategy to image protein-protein interactions that occur in the cell nucleus through an inducible yeast two-hybrid (IY2H) strategy. In Aim 2 we refine an alternate approach referred to as an inducible split reporter (ISR) strategy which is capable of measuring protein-protein interactions in many locations within a cell. In Aim 3 we test the imaging strategies developed in Aims 1 and 2 using new approaches being used to target the interaction of heat shock protein 90 (Hsp90) with p23 using geldanamycin analogs in tumor therapy models in living mice. Finally, in Aim 4 we develop transgenic models to further test methods for imaging protein-protein interactions in deep tissues of living mice. In all aims, care is taken to develop generalizable approaches that should be applicable to other studies using multimodality reporter gene approaches. The significance of the proposed work is it will help to develop and validate new technologies focused on imaging intracellular communication networks. This should lead to many applications including methods to image the interaction of drugs designed to inhibit protein-protein interaction in living subjects. More rapid methods to validate and translate pre-clinical models for cancer therapeutics into the clinic will likely result.